PROJECT SUMMARY Bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development is the most common morbidity complicating preterm birth, and a predictor of poor neurodevelopmental outcomes. Currently it is unclear to what extent lung injury contributes to neurodevelopmental impairment (NDI) in these preterm infants. Based on our preliminary data which demonstrate that circulating exosomes derived from preterm infants who develop severe BPD induce neural stem cell (NSC) death in vitro, the major goal of this proposal is to determine the molecular mechanisms by which these circulating exosomes mediate lung to brain crosstalk and their contribution to brain injury and NDI. Understanding these mechanisms is fundamental to public health as more preterm infants are surviving, yet there remains no effective therapy for either BPD or its associated NDI. Pyroptosis is a newly described form of inflammatory cell death that is solely regulated by gasdermin D (GSDMD), an inflammasome-activated membrane ?pore-forming? protein. Exosomes are nano- sized extracellular vesicles that play a key role in inter-organ communications. We recently demonstrated that preterm infants who develop severe BPD have increased expression of GSDMD and surfactant protein C (SPC), a marker of alveolar type II epithelial cells (AEC), in their circulating exosomes at 1 week of age. In preclinical studies with an experimental model of BPD, similar GSDMD-containing exosomes were isolated from the serum of newborn rats exposed to hyperoxia. Importantly, adoptive transfer of these exosomes into the circulation of normal newborn rats impaired brain development. These findings lead us to propose a novel model that links BPD, exosomal GSDMD and brain injury. The central hypothesis of this model is that GSDMD-containing exosomes are released from AEC into the circulation during the evolving stage of BPD, and neuronal uptake of these exosomes results in neural cell pyroptosis and NDI. This hypothesis will be tested through three aims: Aim 1. To characterize the relationship between GSDMD expression profiles in AEC-derived circulating exosomes and the severity of BPD in preterm infants. Aim 2. To determine the mechanistic functions of circulating exosomes from preterm infants in inducing brain injury and NDI. Aim 3. To elucidate the molecular mechanisms by which hyperoxia stimulates AEC to release GSDMD+ exosomes that induce brain injury and NDI. These studies will delineate a novel exosomal GSDMD-mediated lung to brain crosstalk that is critical in the pathogenesis of brain injury and NDI in premature infants. Completion of this project will provide a strong foundation to further explore therapeutic interventions that target exosomal GSDMD to improve long-term respiratory and neurodevelopment outcomes in these infants. This proposal is aligned with the overarching goal of the NICHD and NHLBI that is to improve children?s health through cutting edge research and it will be executed by a team approach with experts in clinical and basic respiratory research, neuropathology, and exosome and GSDMD biology.